Transposition of the Complementary Developing Solvent Technique to Flash Chromatography: A Structured Step‐Based Fractionation Strategy for Complex Plant Extracts

The Complementary Developing Solvent technique, originally developed for high-performance thin-layer chromatography, employs solvent systems with distinct polarity ranges to achieve enhanced chromatographic resolution while covering a broad chemical space. This study demonstrates the successful adaptation of low polarity developing solvent and high polarity developing solvent systems to normal-phase flash chromatography for systematic fractionation. The system suitability test used in high-performance thin layer chromatography, namely the Universal HPTLC Mix, enabled direct comparisons between high-performance thin layer chromatography and flash chromatography, demonstrating a conserved polarity-driven retention hierarchy across chromatographic scales. To confirm compound identities and elution order, each chromatographic peak was collected and analyzed by tandem mass spectrometry, with MS/MS spectra manually matched against reference standards. Quantitative chromatographic performance was benchmarked using effective peak capacity (Pc), yielding a value of 16.41 for the high-polarity developing system, indicative of a globally balanced separation. In addition, a systematic isocratic step fractionation strategy was implemented by segmenting each elution gradient (low polarity developing solvent and high polarity developing solvent) into seven predefined polarity windows. This strategy was applied on a crude methanolic extract of Sideritis scardica Griseb., confirming robust and reproducible fractionation boundaries in a complex botanical matrix. Collecting a single fraction per isocratic step significantly reduces the number of samples for subsequent analysis such as bioassays and untargeted metabolomic profiling, thereby accelerating natural product discovery and dereplication workflows.